Development of direct observation method of bio-molecules by using environmental phase plate transmission electron microscopy

我们一直在开发一种环境电子显微镜(E-TEM)和相板电子显微镜(P-TEM)相结合的方法，可以直接观察水溶液中生物分子结构。实验表明，这种组合方法可以对水层中的生物分子进行成像，而不会产生任何污渍。本研究获得的空间分辨率约为10 nm。这应该通过使用能量过滤来改善。样品在水中的图像对比度比在真空中的图像对比度降低。模型计算考虑了样品周围水层散射引起的光束展宽、强度降低和背景增加的影响，结果表明，水层厚度的增加降低了图像的对比度、强度和分辨率。有很多孔的塑料薄膜被用来形成碳胶囊。样品和实验溶液被包裹在由间隔物和两个碳膜制成的胶囊中。通过改变制备塑料间隔物的条件，可以控制水层的厚度。与我们以前的可以在样品周围循环气体的环境单元相比，图像质量得到了改善，使用松散凝聚的DNA获得了2 nm的分辨率。

We have been developing a combination method for environmental TEM (E-TEM) and phase-plate TEM (P-TEM) that enables direct observations of the structure of biological molecules in aqueous solution. It is clearly demonstrated that the biological molecules in a water layer can be imaged by the combined method without any stain. The spatial resolution obtained in the present study was about 10 nm. This should be improved by using energy filtering. The image contrast of the specimen in water was reduced in comparison with that in vacuum. A model calculation that includes the effects of beam broadening, intensity decrease, and background increase caused by scattering from the water layer around the specimen shows that an increase in the thickness of the water layer reduces the contrast, intensity, and resolution of the image.A carbon sandwitch environmental cell for wet specimen was developed for environmental transmission electron microscopy. A plastic film with many holes was used to form carbon capsules. The samples and experimental solution are enclosed in the capsules made of the spacer and two carbon films. The thickness of water layer can be controlled by changing conditions to prepare the plastic spacers. The quality of the images was improved comparing with our previous environmental cell which can circulate gas around samples and2nm resolution was obtained by using loosely condensed DNAs.